Protein kinase npk-110

ABSTRACT

The present invention relates to a DNA sequence encoding a neuronal protein kinase (NPK) which phosphorylates tau proteins as well as other microtubule associated proteins (MAPs) in positions crucial for the binding to microtubules. The invention further relates to Serine or Theorine residues and epitopes comprising said residues phosphorylated by said NPK on said MAPs, to antibodies specifically binding to said protein kinase pharmaceutical compositions comprising inhibitors to said protein kinase, in particular for the treatment of Alzheimer&#39;s disease and cancer, to diagnostic kits and to in vitro diagnostic methods for the detection of Alzheimer&#39;s disease and cancer

[0001] The present invention relates to a DNA sequence encoding a novelneuronal protein kinase (NPK) which phosphorylates tau proteins as wellas other microtubule associated proteins (MAPs) in positions crucial forthe binding to microtubules. The invention further relates to Serine orTheorine residues and epitopes comprising said residues phosphorylatedby said NPK on said MAPs, to antibodies specifically binding to saidprotein kinase, pharmaceutical compositions comprising inhibitors tosaid protein kinase, in particular for the treatment of Alzheimer'sdisease and cancer, to diagnostic kits and to in vitro diagnosticmethods for the detection of Alzheimer's disease and cancer.

[0002] Microtubule associated proteins (MAPs) regulate the extensivedynamics and rearrangement of the microtubule network which is thoughtto drive neurite outgrowth (reviewed recently by Hirokawa, 1994).Several lines of evidence suggest that the phosphorylation state ofMAPs, balanced by protein kinases and phosphatases in a hitherto unknownway, plays a pivotal role in the modulation of these events. Tauprotein, a class of MAPs in mammalian brain (Cleveland et al., 1977), isphosphorylated on several sites in vivo (Butler & Shelanski 1986;Watanabe et al., 1993) and is a substrate for many protein kinases invitro (reviewed by Lee, 1993; Goedert, 1993; Mandelkow & Mandelkow,1993; Anderton, 1993). During neuronal degeneration in Alzheimer'sdisease, tau protein aggregates into paired helical filaments (PHFs),the principal fibrous component of the characteristic neurofibrillarylesions (reviewed by Lee & Trojanowski, 1992). Tau isolated from theseaggregates displays some biochemical alterations, of whichhyperphosphorylation is the most striking (Grundke-Iqbal et al., 1986;Brion et al., 1991; Ksiezak-Reding et al., 1992; Goedert et al., 1992).Most of the reported aberrant phosphorylation sites are Ser/Thr-Prosequences (Lee et al., 1991; Biernat et al., 1992; Lichtenberg-Kraag etal., 1992; Gustke et al., 1992; Watanabe et al., 1993), suggesting adysregulation of proline-directed kinases (Drewes et al., 1992;Mandelkow et al., 1992; Hanger et al., 1992; Vulliet et al., 1992;Baumann et al., 1993; Paudel et al., 1993, Kobayashi et al., 1993) orthe corresponding phosphatases (Drewes et al., 1993; Gong et al., 1994).Phosphorylation-dependent antibodies, which discriminate between‘normal’ tau and the hyperphosphorylated, ‘pathological’ forms, wereprepared by several laboratories (Kondo et al., 1988; Lee et al., 1991;Mercken et al., 1992; Greenberg et al., 1992). All of these antibodieswere shown to be directed against epitopes of the Ser/Thr-Pro type (Leeet al., 1991; Biernat et al., 1992; Lichtenberg-Kraag et al., 1992; Langet al., 1992; Watanabe et al., 1993).

[0003] The microtubule binding region of tau (FIG. 1) includes three orfour pseudorepeats of 31 residues each depending on isoform type (Lee etal., 1989; Goedert et al., 1989; Himmler et al., 1989). This regionprobably forms the building block of the paired helical filaments (Kondoet al., 1988; Wischik et al., 1988; Ksiezak-Reding & Yen, 1991; Wille etal., 1992). It does not contain any of the 14-16 Ser/Thr-Pro motifs,which accumulate in the regions flanking the repeats. However, itcontains a conserved Serine residue (Ser262) within the sequence KIGS inthe first repeat, which was found to be one of the predominant sitesphosphorylated by a tissue extract from brain (Gustke et al., 1992).This site is also found to be phosphorylated in Alzheimer PHF-tau, butnot in ‘normal’ tau or fetal tau (Hasegawa et al., 1992). So far, it isthe only pathological phosphorylation site found within the repeatdomain of tau.

[0004] Recently, a site-directed mutagenesis approach was used to showthat phosphorylation of tau at this site strongly decreases itsmicrotubule binding capacity, whereas the phosphorylation on Ser/Thr-Promotifs had only a minor effect (Biernat et al., 1993). This initiated asearch for protein kinases in neuronal tissue with the ability tophosphorylate tau at Ser262. The technical problem underlying thepresent invention was to provide a protein kinase which is causative forthe onset of Alzheimer's disease by phosphorylating the crucial Serine262 residue of human tau protein and a corresponding nucleotidesequence.

[0005] The solution to this technical problem is achieved by providingthe embodiments characterised in the claims.

[0006] Thus, the present invention relates to a DNA sequence encoding aneuronal protein kinase (NPK) or a functional fragment thereof that iscapable of phosphorylating a sequence motive of the type KXGS in tau,MAP4, MAP2 and MAP2c characterised by the following features:

[0007] (a) it encodes the amino acid sequence depicted as MARK-1 inTable 6;

[0008] (b) it encodes the amino acid sequence depicted as MARK-2 inTable 6; or

[0009] (c) it hybridises to the DNA of (a) or (b).

[0010] The term “DNA sequence” comprises any DNA sequence such asgenomic or cDNA, semisynthetic or synthetic DNA.

[0011] It was surprisingly found that none of the prior art kinases ismediating the phosphorylation of the four KXGS motifs in the repeatdomain of tau to an extent that is sufficient to explain the biologicaland pathological effects associated with said phosphorylation. This isparticularly true-for Serine residue 262 which is indicative of theonset of Alzheimer's disease. Instead, the present invention provides aDNA sequence encoding a novel protein kinase with the above identifiedfeatures which is responsible for the phosphorylation of the amino acidresidues crucial for the onset of Alzheimer's disease. Said proteinkinase is, also termed NPK, MARK-1 or MARK-2 throughout thisapplication. The numbering of amino acid residues referred to in thisapplication ensues with regard to the sequence of htau 40, the longestof the human tau isoforms (441 residues, Goedert et al., 1989).

[0012] In a preferred embodiment, the present invention further relatesto a DNA sequence wherein the neuronal protein kinase (NPK) ischaracterised by the following features:

[0013] (a) it has an apparent molecular weight of 110 kD as determinedby SDS-PAGE;

[0014] (b) it phosphorylates Serine residues 262, 293, 305, 324 and 356of human tau protein; and

[0015] (c) it comprises the following amino acid sequencesKLDTFCGSPPYAAPELFQGK DRWMNVGHEEEELKPYAEP (K) SSRQNIPRCRNNI

[0016] In a preferred embodiment of the DNA sequence of the presentinvention, the NPK is further characterised by the following features:

[0017] (d) it is deactivated by phosphatase PP-2A; and

[0018] (e) it phosphorylates the following Serine or Threonine residuesof tau related microtubule-associated proteins (MAPs) MAP2, MAP2c andMAP4

[0019] MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796, S1799

[0020] MAP4: T829, T873, T874, T876, S899, S903, S928, S941, S1073

[0021] (f) it causes the dissociation of tau, MAP4, MAP2 and MAP2c frommicrotubules.

[0022] Another surprising finding that was made in accordance with thepresent invention is that the NPK by phosphorylatingmicrotubule-associated proteins other than tau causes dissociation ofthese proteins from microtubules. This in turn results in thedestabilisation of said microtubules, an increased dynamic instabilitythereof, and the ensuing effects on cell proliferation, celldifferentiation, or cell degeneration. The NPK of the invention thus hasthe capacity to regulate the dynamics and rearrangements of microtubulesin brain via the phosphorylation of tau or other MAPs. The findingreferred to above has important implications for the role in the kinaseof the invention in the generation of cancer.

[0023] This is because it is believed that cancer essentially isuncontrolled cell proliferation. Many anti-cancer drugs thereforeinterfere with cellular division and proliferation by poisoning themicrotubules. On the other hand, “oncogenes” are often kinases, thecellular regulation of which is impaired. The deregulation of a kinaseequal or homologous to the NPK of the invention could have seriouseffects on the stability of microtubules of various cell types. Asmicrotubules play an important role in cell division, deregulation ofsaid NPK can in turn lead to an uncontrolled cellular division and thetransformation of normal cells to cancer cells. Alternatively, thederegulation of said NPK could provide postmitotic terminallydifferentiated cells such as neurons (which do not divide) with astimulus to divide. This “unnormal” stimulus would lead the neuronsdirectly into apoptosis (and thus, an Alzheimer's like state) becausedue to their differentiation status they are unable to divide.

[0024] In a further preferred embodiment of the DNA sequence of thepresent invention, the NPK is obtainable from brain tissue by thefollowing steps:

[0025] (a) homogenisation of brain extract and subsequent centrifugationthereof;

[0026] (b) chromatography of the supernatant obtained in step (a) oncellulosephosphate, wherein the NPK active fractions elute between 200to 400 mM NaCl;

[0027] (c) ammonium sulfate precipitation of active fractions obtainedin step (b) and dialysis of the precipitate;

[0028] (d) anion exchange chromatography of the dialysate obtained instep (c) on Q-Sepharose (Pharmacia) and elution of the NPK activefractions, wherein said NPK active fractions elute as a single peak atabout 0.2 M NaCl, with subsequent dialysis of the active fractions;

[0029] (e) cation exchange chromatography on Mono S HR 10/10(Pharmacia);

[0030] (f) chromatography on Mono Q HR 5/5, wherein the NPK activefractions elute at about 250 mM NaCl;

[0031] (g) gel filtration chromatography on Superdex G-200, wherein theNPK activity elutes with an apparent molecular weight of 100 kD; and

[0032] (h) affinity chromatography on ATP-cellulose, wherein the NPKactive fractions elute with an apparent molecular weight of about 110 kDas determined by SDS-PAGE;

[0033] wherein the NPK activity is measured by incubating a peptidecomprising amino acid residues 255 to 267 of human adult tau in thepresence of radioactively labelled ATP and determining the radioactivityincorporated into said peptide.

[0034] Further details as to how this NPK of the invention which in oneembodiment has an apparent molecular weight of 110 kD (NPK-110) can beisolated are provided in Example 1. However, the person skilled in theart would know from the technical teaching given above how to supplementsaid details.

[0035] The NPK of the invention may be derived from any vertebratebrain. In a preferred embodiment, the NPK is derived from a mammalianbrain.

[0036] The invention also relates to a RNA sequence complementary to theDNA sequence of the invention.

[0037] In a particularly preferred embodiment, said mammalian brain ishuman or porcine brain.

[0038] The invention further relates to a polypeptide encoded by the DNAsequence or a functional fragment or derivative thereof. Saidpolypeptide, fragment or derivative may be posttranslationally orchemically modified. Throughout this specification, the term NPK or,alternatively, MARK (1 or 2) may also comprise such fragments orderivatives, even if this is not specifically indicated.

[0039] The present invention further relates to the following Serine orThreonine residues phosphorylated by NPK-110of tau relatedmicrotubule-associated proteins (MAPs) MAP2, MAP2c and MAP4:

[0040] MAP2/MAP2c: S37, S1536,S1676, S1707, S1792, S1796, S1799

[0041] MAP4: T829, T873, T874, T876, S899, S903, S928, S941, S1073

[0042] and to epitopes comprising said phosphorylated Serine orThreonine residues.

[0043] The invention relates further to an antibody specifically bindingto the NPK of the invention.

[0044] Said antibody may be a serum derived or a monoclonal antibody.The production of both monoclonal and polyclonal antibodies to a desiredepitope is well known in the art (see, for example, Harlow and Lane,Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor, 1988). Furthermore, said antibody may be a natural or anantibody derived by genetic engineering, such as a chimeric antibodyderived by techniques which are well understood in the art. Moreover,the term antibody as used herein also refers to a fragment of anantibody which has retained its capacity to bind the specific epitope,such as a Fab, F(ab)₂ or an Fv fragment.

[0045] Additionally, the present invention relates to an antibodyspecifically binding to epitopes comprising the phosphorylated Serine orThreonine residues of MAP2, MAP2c and MAP4:

[0046] MAP2/MAP2C: S37, S1536, S1676, S1707, S1792, S1796, S1799

[0047] MAP4: T829, T873, T874, T876, S899, S903, S928, S941, S1073

[0048] Again, said antibody may be a polyclonal or a monoclonalantibody, or a fragment thereof retaining its binding specificity.

[0049] In a preferred embodiment, the antibody of the invention is amonoclonal antibody or a fragment or derivative thereof.

[0050] In a further preferred embodiment of the invention, said antibodyis a polyclonal antibody or a fragment or a derivative thereof.

[0051] The invention furthermore relates to a pharmaceutical compositionwhich contains a specific inhibitor of the NPK of the invention,optionally in combination with a pharmaceutically acceptable carrierand/or diluent.

[0052] The term “specific inhibitor of the NPK of the invention” refersto substances which specifically inhibit the enzymatic action of theprotein kinase of the present invention. Inhibitors to enzymes such asprotein kinases and their mode of action are well known in the art. Forexample, such an inhibitor may bind to the catalytic domain of theenzyme, thus rendering it incapable of converting its substrate.

[0053] Said pharmaceutical composition may be administered to a patientin need thereof by a route and in a dosage which is deemed appropriateby the physician familiar with the case. Pharmaceutically acceptablecarriers and/or diluents are well known in the art, and may beformulated according to the route of administration or the specialdisease status of the patient.

[0054] In a preferred embodiment, the present invention relates to apharmaceutical composition for the treatment of Alzheimer's disease.

[0055] Again, said pharmaceutical composition may be administered to apatient in need thereof by a route and in a dosage which is deemedappropriate by the physician handling the case.

[0056] In a further preferred embodiment, the pharmaceutical compositionof the present invention is used for the treatment of cancer.

[0057] As has been pointed out above, the deregulation of the NPK of theinvention can lead a variety of cell types expressing microtubuleassociated proteins into a pathway that eventually results in theneoplastic transformation of said cells. Accordingly, a pharmaceuticallyeffective amount of an NPK inhibitor will halt and/or reverse thetransformation process. The amount of inhibitor to be administered willbe determined by-the physician handling the respective cases.

[0058] In a further preferred embodiment of the pharmaceuticalcomposition of the invention, said inhibitor is the antibody of theinvention, a phosphatase capable of dephosphorylating the NPK of theinvention, preferably phosphatase PP-2A, an inhibitor of the activatingkinase of said NPK, a tau derived peptide comprising the Ser262 residueor a MAP2, 2c or MAP4 derived peptide comprising at least one of theSerine or Threonine residues of MAP2, MAP2c or MAP4:

[0059] MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796, S1799

[0060] MAP4: T829, T873, T874, T876, S899, S903, S928, S941, S1073

[0061] The terms “tau derived peptide comprising the Ser262 residue anda MAP2, 2c or MAP4 derived peptide comprising at least one of the Serineor Threonine residues of MAP2, MAP2c and MAP4:

[0062] MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796, S1799

[0063] MAP4: T829, T873, T874, T876, S899, S903, S928, S941, S1073”

[0064] as used herein refers to a peptide which in its three dimensionalstructure reconstitutes the natural conformation of the tau protein orthe MAP2, 2c or 4 proteins with regard to the epitope comprising Serineresidue 262 (tau) or the other residues referred to above (MAP) MAP2,MAP2c and MAP4. These peptides will mimic the natural substrate (i.e.tau or tau related MAPs) of the NPK of the invention, but will notdisplay any NPK associated biological effect. The synthesis of saidpeptides which solely may consist of the epitopes, or may compriseadditional flanking amino acids, is well known in the art.

[0065] The present invention further relates to a diagnostic compositioncomprising:

[0066] (a) the NPK of the invention;

[0067] (b) the antibody or fragment or derivative of the invention;and/or

[0068] (c) a peptide comprising the phosphorylatable Serines orThreonines of tau, MAP2, MAP2c or MAP4 indicated above.

[0069] Said diagnostic composition may, for example, be used for thedetection of Alzheimer's disease or cancer or the onset thereof. Theantibody of the invention may be used to detect abnormal, in particularhigher concentrations or levels, of the NPK of the invention, a higherdegree of activation of said NPK, which are indicative of said diseases.The NPK delivered with the composition could be used as an internalcontrol. On the other hand, the above defined peptides may be used assubstrates to detect an abnormal activity of the NPK of the invention.Again, the activity of the NPK comprised in the diagnostic compositionmay serve as an internal control.

[0070] The antibody specifically binding to the phosphorylated Serineresidues enumerated above and comprised in MAP4, MAP2 or MAP2c may beused to detect an abnormal phosphorylation status or pattern of thesemicrotubule associated proteins which is indicative of cancer.

[0071] Further applications of the diagnostic composition are asfollows. Thus, in one embodiment, said diagnostic composition maycomprise an antibody of the invention directed to one of the epitopesreferred to above. For example, an Alzheimer's or cancer correlateddisease state of a sample may be detected by treating said sample withan antibody recognising one or more of said epitopes. Theantibody-epitope (hapten) complex may be visualised using a secondantibody directed to the antibody of the invention and being labelledaccording to methods known in the art (see, for example, Harlow andLane, ibid.).

[0072] In still another embodiment of the present invention, saiddiagnostic composition may consist of an epitope referred to above andan antibody of the invention. Treatment of a sample with said antibodymay give rise to conclusions with regard to the disease state of thecorresponding patent, if the binding of said antibody to said sample isbrought in relation to binding of said antibody to said epitope referredto above used as a reference sample.

[0073] In still another embodiment, the diagnostic composition maycomprise an epitope referred to above, the NPK of the invention and anantibody of the invention. Kinase activity may be monitored with respectto phosphorylation of the sample as compared to the phosphorylation ofthe epitope of the invention. From the quantitated NPK activity thephosporylation state of the tau protein or the MAP2, 2c or 4 containedin said sample and therefore the disease state of the patient may bededuced. The kinase activity may, for example, be deduced by including asubstrate analog in the same reaction, which is visually detectable uponenzymatic conversion. Such substrate analogs are widely used in the art.Alternatively, the amount of a phosphorylated tau protein or MAP2, 2c or4 in the sample may be detected after treatment with the kinase of theinvention by employing an antibody of the invention directed to thephosphorylated epitope and using the amount of antibody-epitope complexprovided by the diagnostic composition as an internal standard, or bydetermining the amount of phosphate incorporated into tau protein orMAP2, 2c or 4 by the NPK, for example, by radioactive tracer methodswhich are well known in the art.

[0074] It should be kept in mind, however, that the person skilled inthe art, being familiar with diagnostic principles, can easily combinethe above mentioned compound in a different manner or supplement thecomposition with secondary or tertiary, labelled or unlabelledantibodies, or with enzymes and substrates. These embodiments are alsocovered by the present invention.

[0075] In still another embodiment, the invention relates to a methodfor the in vitro diagnosis and/or monitoring of Alzheimer's diseasecomprising assaying a cerebrospinal fluid isolate of patient or carryingout a biopsy of nerve tissue (for example, olfactory epithilium) andtesting said tissue for the presence of the NPK of the invention.

[0076] The invention further relates to a method for the in vitrodiagnosis and/or monitoring of Alzheimer's disease comprising assaying acerebrospinal fluid isolate of a patient or carrying out a biopsy ofnerve tissue and testing said tissue for the presence of unphysiologicalamounts of the NPK of the invention, or for unphysiological activity ofsaid NPK.

[0077] An example of a nerve tissue suitable for said biopsy is theolfactory epithelium.

[0078] The method of the invention may, for example, be carried out byusing the diagnostic composition of the invention, in particular theantibody directed to said NPK. Therefore, in a preferred embodiment ofthe invention, the NPK of the invention is detected by the antibody ofthe invention specifically binding to said NPK.

[0079] Additionally, the invention relates to a method for the in vitrodiagnosis for cancer or the onset of cancer comprising assaying asuitable tissue or body fluid for the presence of phosphorylated Serineor Threonine residues of tau related microtubule associated proteins(MAPs) MAP2, MAP2c and MAP4 in the positions:

[0080] MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796, S1799

[0081] MAP4: T829, T873, T874, T876, S899, S903, S928, S941, S1073”

[0082] or for the presence of unphysiological amounts of the NPK of theinvention or an NPK specific phosphatase. It is understood that thephosphorylation status of the Serine or Threonine residues has to be anunphysiological one. Methods for determining such a phosphorylationstatus have been described in detail in PCT/EP 92 02 829, which isincorporated herein by reference.

[0083] The assay for said phosphorylated Serine or Threonine residuesmay, for example, be carried out using an antibody specificallydetecting said phosphorylated residues or the epitopes comprising saidresidues.

[0084] The amount of the NPK in the sample may be measured by usingantibodies specifically directed thereto or by measuring their activityusing a suitable substrate, for example, a peptide comprising the abovereferenced Serine or Threonine in a non-phosphorylated state or any ofMAP2, MAP2c and MAP4 in unphosphorylated state. Methods for measuringthe phosphorylation status of proteins have been described in detail inPCT/EP 92 02 829. The activity of the phosphatases, for example PP-2A,PPI or calcineurin may be tested by providing the substrate, NPK of theinvention, for example, comprised in the diagnostic composition of theinvention.

[0085] A suitable tissue or body fluid for carrying out this in vitromethod of the invention is cerebrospinal fluid, blood, biopsies oftissue (for example, liver or skin).

[0086] Still another object of the invention is to provide a method forthe in vitro conversion of normal MAP2, MAP2c or MAP4 by the treatmentwith the NPK of the invention into proteins phosphorylated at positions:

[0087] MAP2/MAP2c: S37, S1S36, S1676, S1707, S1792, S1796, S1799

[0088] MAP4: T829, T673, T874, T876, S899, S903, S928, S941, S1073”

[0089] said phosphorylation status being indicative of cancer or theonset of cancer. The conditions allowing the phosphorylation of saidMAPs can be determined by following the general teachings provided bythe present application. The phosphorylated MAPs can then be recognisedby specific antibodies. The results of said in vitro method will allowfurther insights into the generation of cancer.

[0090] Moreover, inhibitors may be tested which prevent the conversionof normal to MAP protein phosphorylated in the positions indicatedabove. These “inhibitors” may be specific for the epitope to bephosphorylated by, for example, blocking the epitope, or may be directedto various domains on the protein kinase of the invention, NPK, as longas they prevent or disturb its biological activity. Another type ofinhibition is the antagonistic action of phosphatases on said MAPs orsaid NPK, or the inhibition of the activating kinase of said NPK.Furthermore, the MAP generated by the method of the present inventionmay be employed in binding studies to microtubule structures in vitroand in vivo, thus contributing to the elucidation of the molecular basisunderlying cancer.

[0091] The present invention relates, moreover, to the use of thephosphorylated Serine or Threonine residue(s) of the MAP of theinvention or the epitope comprising said residue(s) for the generationof specific antibodies indicative of cancer or the onset of cancer.

[0092] The methods for obtaining said antibodies are well known in theart; thus, the generation of polyclonal or monoclonal antibodies may beconducted using standard methods (see, for example, Harlow and Lane,ibid.). If an oligo- or polypeptide is used for the generation ofantibodies, it is desirable to couple the peptide comprising the epitopeto a suitable carrier molecule capable of inducing or enhancing theimmune response to said epitope, such as bovine serum albumin or keyholelimpet hemocyanin. The methods of coupling hapten (comprising or beingidentical to the epitope) and carrier are also well known in the art(Harlow and Lane, ibid.). It is also to be understood that any animalsuitable to generate the desired antibodies may be used therefor.

THE FIGURES SHOW

[0093]FIG. 1: Bar diagram of tau (isoform htau40, the largest one incentral nervous tissue, Goedert et al., 1989), construct K18 containingthe four repeats, and several sites phosphorylated by the kinaseactivity from brain (Gustke et al., 1992). The hatched boxes near theN-terminus are inserts which may be absent because of differentialsplicing, the boxes labelled 1-4 represent the four repeats, of whichrepeat 2 may be absent. Most phosphorylated sites are in Ser-Pro orThr-Pro motifs outside the repeats, but the brain kinase activity alsophosphorylates two sites within the repeats, Ser262 and Ser356.

[0094]FIG. 2 Isolation of NPK110 from porcine brain. (A) The tissueextract was loaded onto phosphocellulose and eluted stepwise with 0.15-1M NaCl. The filled bars show the total protein concentration of theeluted material, open bars show the activity as measured with tauconstruct K18 as substrate. (B) The material eluted with 0.35-0.5 M NaClwas submitted to ammonium sulfate precipitation and the precipitatedialysed and loaded onto a Q-Sepharose column. The closed symbols showthe protein concentration, open symbols the activity profile. Thegradient composition is indicated on the right axis. (C) Fractions 8-15from Q-Sepharose were dialysed and loaded onto a SP-Sepharose column.(D) Fractions 12-16 from SP-Sepharose were dialysed and loaded onto aMono Q HR 5/5 column. (E) Fractions 9-11 from Mono Q were loaded onto aSuperdex 200 gel filtration column. The elution positions of molecularweight markers are indicated on the right axis.

[0095]FIG. 3: Final purification of NPK110 by affinity chromatography onATP-Sepharose (SDS PAGE, lanes 1-3) and characterisation by in-gelphosphorylation (autoradiography, lanes 4-6). The most active fractionsfrom the gel filtration column (lane 1) were loaded onto an ATP affinitycolumn. The kinase was eluted specifically with 5 mM ATP (lanes 2, 3).The silver stained gel shows a fuzzy band with an apparent molecularweight of approximately 110 kDal and a second, sharp band with 95 kDal.Lanes 4-6 show autoradiograms of the in-gel phosphorylation of thesamples in lanes 1-3. As a substrate, tau (5 μM) was polymerised intothe gel matrix. After renaturation and incubation with g-³²P ATP, it isclearly shown that only the 110 kDal band displays kinase activitytowards tau.

[0096]FIG. 4: Phosphorylation of wild type tau and construct K18(microtubule binding domain) by NPK110. Htau40 (10 μM, lanes 1, 2) andK18 (20 μM, lanes 3, 4) were phosphorylated with 5 μU/ml of NPK110 and 2mM g-³²P-ATP at 37° C. for 2 hours. Aliquots were electrophoresed on a7-20% SDS gradient gel: Lanes 1, 2, htau40 before and afterphosphorylation, lanes 3, 4, K18 before and after phosphorylation. Notethe small molecular weight shift upon phosphorylation in lanes 2 and 4.The right side shows an autoradiograph of the same gel; phosphorylatedhtau40 and K18 are seen in lanes 2 and 4.

[0097]FIG. 5: Tryptic phosphopeptide maps of wild type tau (htau40) andconstruct K18 phosphorylated with NPK110. 30 μg of tau werephosphorylated with 0.5 μU NPK110 for 2 h at 37° C. (A) full length4-repeat tau (htau40), (B) construct K18 (MT binding region, residues244-372 of full length tau), (C) diagram of the more prominent spots:Spot 1 on upper left contains Ser262, spot 2 on upper right Ser356, spot3 (below 1) Ser305, spot 4 (always part of an overlapping doublet)contained Ser324, spot 5 Ser293 (this tryptic peptide CGSK was notrecovered from the HPLC column, presumably because of its small size,but the spot could be identified by site-directed mutagenesis). (D)Mixture of identical amounts of counts (10,000 cpm) derived fromphosphopeptides shown in (A) and (B). The identification ofphosphorylation sites shown in (C) was performed by two dimensionalanalysis of the HPLC-purified and sequenced peptides (listed in Table1). 10,000 cpm of the purified peptides each were analysed alone and incombination with a 5000 cpm aliquot of the phosphopeptides shown in (A)in order to allow unambiguous identification.

[0098]FIG. 6: Phosphorylation of Ser262 abolishes the binding of tau tomicrotubules. (A) Binding of tau to taxol-stabilised microtubules (30μM) was measured in a cosedimentation assay as described below inExample 2. Full length wild-type tau (‘wt’, htau40) and a Ser262 to Alamutant (A262) (10 μM) were previously phosphorylated with NPK110(finalconcentration 8.5 μU/ml) for 2 hours at 37° C. Curves were obtained bynon-linear regression (Biernat et al., 1993).

[0099] The binding of wild-type tau is completely abolished byphosphorylation (closed circles), whereas the A262 mutant still binds,although with lower affinity (triangles). For comparison, the binding ofunphosphorylated tau is also shown (open circles).

[0100] (B) Microtubule-bound tau comes off during phosphorylation byNPK110. htau 40 (10 μM) was incubated with taxol-stabilised microtubules(30 μM). At t=0, NPK110was added to a final concentration of 10 μU/ml,and aliquots were withdrawn at time intervals from one to 20 hours andpelleted. Tau was measured in the pellets and supernatants bydensitometry of the SDS gels - (closed circles). Incorporated phosphatewas measured by Cerenkov counting of gel pieces (open circles) and isindicated on the right axis. Phosphate incorporation in tau withoutmicrotubules is shown to proceed faster (squares).

[0101]FIG. 7: Dark field video microscopy of microtubules and effect ofphosphorylation of Ser262 on tau. Microtubules (5 μM tubulin) werenucleated on sea urchin sperm axonemes in the presence of 2.5 μM tau(isoform htau40) and 10 μU/ml of NPK110. A, 20 min without ATP, B, withATP. In A the microtubules grow continuously, in B Ser262 can bephosphorylated, leading to a destabilisation and shortening ofmicrotubules. Bar=10 μm.

[0102]FIG. 8: Effect of the unphosphorylated and NPK110-phosphorylatedtau on the length of axoneme nucleated microtubules measured bydarkfield microscopy. For each condition 500 to 600 microtubule plusends were measured; the mean length was plotted against time. Tubulinconcentration was 5 μM; note that without added tau, no microtubules areobserved at this concentration. Tau was 2.5 μM in all cases. In controlexperiments, ATP was omitted (‘- ATP’).

[0103] (A) Tau pre-phosphorylated by NPK110 does not promote microtubulegrowth (filled circles) but the pre-phosphorylated point mutant A262does (triangles, in accordance with time resolved binding assay in FIG.6B).

[0104] (B) Tubulin and tau were mixed at 4° C. with 10 μU/ml of NPK110(final concentration) in the presence (closed circles) or absence (opencircles) of 2 mM Mg-ATP. At t=0, the temperature was raised to 37° C.With wild type tau and no ATP, microtubules grow continuously (opencircles); the same result is obtained with the mutant Ser262-Ala(triangles). However, wild type tau plus ATP leads to initial growth butsubsequent shrinkage (closed circles). (C-E) Microtubule lengthhistograms at 5 min and 30 min of the corresponding curves in B. Eachsample shows a pronounced peak around 20 μm after 5 min (empty circles).If Mg-ATP was absent (C) or Ser262 was mutated into Ala (E) thedistribution became broader and shifted to greater lengths at 30 min. Bycontrast, phosphorylation of tau successfully decreased the meanmicrotubule length within 30 min of incubation (D).

[0105]FIG. 9: Tryptic phosphopeptide maps of wild type tau (htau40) andconstruct K18 phosphorylated with (A) brain extract, (B) NPK110, (C)PKC, or (D) PKA, respectively. The numbering of the spots is analogousto FIG. 5 (spot 1:Ser262, spot 2:Ser356, spot 3:Ser3O5, spot 4:Ser324,spot 5:Ser293). The panels on the right show the correspondingtwo-dimensional phosphoamino acid analysis of full length tau for eachkinase.

[0106]Fig. 10: Diagram representing the influence of differentphosphorylation sites on tau-microtubule interactions. The majority ofSer/Thr-Pro motifs are in the flanking regions of the repeat domain,they have only a small influence on the binding of tau. The repeatdomain contains several phosphorylatable non-Ser-Pro sites, especiallythe four KXGS motifs. Of these, Ser262 in the first KIGS motif has byfar the greatest influence on microtubule binding.

[0107]FIG. 11: Phosphopeptide map of recombinant MAP2c phosphorylated byNPK-110. The peptides contain the following phosphorylated residues:I=Ser1707, II=Ser1676, III=Ser37 and Ser1536, IV=Ser1792, Ser1796 andSer1799

[0108] (numbering of residues following Albala et al., 1993).

[0109]FIG. 12: Phosphopeptide map of MAP4 fusion protein phosphorylatedby NPK-110. The peptides contain the following phosphorylated residues:I=Thr829, II=Ser941, III=Ser928, IV=Thr873, Thr874 and Thr876, V=Ser899and Ser903, VI=Ser1073, VII=Ser928

[0110] (numbering of residues following West et al., 1991).

[0111]FIG. 13: Effect of the unphosphorylated and NPK-110-phosphorylatedMAP4, MAP2 and MAP2c on the length of axoneme nucleated microtubulesmeasured by darkfield microscopy. For each condition 500 to 600microtubule plus ends were measured; the mean length was plotted againsttime. Tubulin concentration was 5 μM; MAPs were 1 μM. Note that withoutadded MAPs, no microtubules were observed at this concentration.

[0112] (a) Tubulin and MAP4 were mixed at 4° C. with 10 μU/ml of NPK-110(final concentration in the presence (closed circles) or absence (opencircles) of 2 mM Mg-ATP. At t=0, the temperature was raised to 37° C.With MAP4 and no ATP, microtubules grow continuously (open circles).However, MAP4 plus ATP leads to initial growth but subsequent shrinkage(closed circles) because MAP4 becomes phosphorylated, detaches frommicrotubules, and microtubules are destabilised.

[0113] (b) Same experiment as in (a) but using MAP2, with similarresults.

[0114] (c) Same experiment as in (a) but using MAP2c, with similarresults.

[0115]FIG. 14: Northern Blot of adult and fetal human tissues with aMARK cDNA probe.

[0116] left: adult tissue

[0117] lane 1: Pancreas (Pa)

[0118] lane 2: Kidney (Ki)

[0119] lane 3: Muscle (Mu)

[0120] lane 4: Liver (Li)

[0121] lane 5: Lung (Lu)

[0122] lane 6: Placenta (P1)

[0123] lane 7: Brain (Br)

[0124] lane 8: Heart (H)

[0125] Right: fetal tissue

[0126] lane 9: Kidney (Ki)

[0127] lane 10: Liver (Li)

[0128] lane 11: Lung (Lu)

[0129] lane 12: Brain (Br)

[0130]FIG. 15: Binding of recombinant wild type MAP2c and MAP2c pointmutants to taxol stabilized microtubules (30 μM tubulin dimers) underthe influence of phosphorylation by p110MARK. Open circles: wild-typeMAP2c, non-phosphorylated. The binding is tight (Kd about 0.25 μM) andsaturates around 17 μM ligand, or about 1 MAP2c molecule per 2 tubulindimers. Closed circles: wild-type MAP2c, phosphorylated previously withp110MARK (2.5 milliUnits/ml; 1 Unit corresponds to 1 μmol of phosphatetransferred to MAP2c per minute at 30° C.) for 2 h. Note that there isessentially no binding. Closed and open squares: MAP2cA319 andMAP2cA350, phosphorylated previously with p110MARK (2.5 milliUnits/ml)for 2 h. In these mutants the serines 319 or 350 in the KXGS motifs inthe first or second repeat were point mutated to alanines. The affinityto microtubules decreases markedly (Kd ≈7 μM) although the stoichiometryremains similar to the wildtype MAP2c. Triangles: MAP2cA319/A350,phosphorylated previously with p110mark (2.5 milliUnits/ml) for 2 h. Inthis mutant both serines 319 and 350 are mutated to alanines. Thebinding is similar to the unphosphorylated protein, showing that thesensitivity to phosphorylation has disappeared because the two KXGSmotifs are no longer phosphorylatable.

[0131]FIG. 16: Effects of unphosphorylated and p110MARK-phosphorylatedMAP4 (A), MAP2 (B), MAP2c (C) and MAP2c point mutants (D) on the lengthof self- nucleated microtubules measured by darkf ield microscopy. Foreach condition 500-800 microtubules were analyzed, and the mean lengthwere plotted against time. Tubulin concentration was 10 μM in all cases,the concentration of MAP4 and MAP2 was 1 μM, that of MAP2c 2 μM. Incontrol experiments, ATP was omitted (‘-ATP’).

[0132] Open circles in A, B and C: The MAPs were preincubated for 30 minwith 2.5 mUnits/ml p110MARK (final concentration), but without ATP. Byadding 10 μM tubulin, microtubules were nucleated and the meanmicrotubule length increased up to about 20 μm within 30 min. Bycontrast, if ATP was present no self-nucleation occurred, showing thatthe phosphorylation of the MAPs prevented microtubule formation. Shortmicrotubules of about 2 um length could only be observed by addingaxonemes (10-100 fM) to promote seeded nucleation (open triangles in A,B, C).

[0133] Closed circles in A, B, and C: Tubulin and MAP were mixed at 4°C. with 2.5 mUnits/ml of p110MARK (final concentration), and thetemperature was shifted immediately to 37° C. (so that initially theMAPs were unphosphorylated). Microtubule growth was promoted in allthree cases, but the final mean microtubule length was only about halfof that observed for the unphosphorylated MAPs (compare open circles).

[0134] D: The effect of phosphorylation site point mutations of MAP2c.All proteins were phosporylated as described above (with 30 minpreincubation). Triangles; wildtype MAP2c, closed circles; MAP2cA319(KXGS in first repeat mutated to KXGA), squares; MAP2cA350 (KXGS insecond repeat mutated to KXGA), closed squares; MAP2cA319/A350 (KXGS inboth repeats mutated to KXGA).

[0135] The Examples illustrate the invention.

[0136] Regarding the tau proteins described in the examples, onlyrecombinant human tau proteins expressed in E. coli were used. cDNAclones were prepared as described by M. Goedert (Goedert et al., 1989)and were expressed using variants of the pET expression vector (Studieret al., 1990). The proteins were purified making use of the heatstability of tau and Mono S FPLC (Hagestedt et al., 1989). Construct K18is derived from the 4-repeat tau isoform and comprises the microtubulebinding region, residues 244 to 372 (Biernat et al., 1993). Mutant‘A262’ is based on the longest human isoform. A single residue, Ser262,was changed into alanine using conventional technology.Phosphocellulose-purified tubulin (PC-tubulin) was prepared from porcinebrain following Mandelkow et al., 1985. Protein kinase A catalyticsubunit (isolated from bovine heart, activity 27 catalytic subunit(isolated from bovine heart, activity 27 nU/μl based on kemptide, 100pU/μl based on casein) was obtained from Promega, Protein kinase C(isolated from rat brain, activity 80 pU/μl based on histone H1) wasfrom Boehringer Mannheim.

EXAMPLE 1

[0137] Purification and characterisation of the protein kinase NPK110.

[0138] All operations were performed at 4° C. Fresh porcine brains(approx. 1 kg) were obtained at the local slaughterhouse and homogenisedinto 1 litre of buffer A (50 mM Tris, pH 8.5, containing 5 mM EGTA, 100mM NaF, 1 mM PMSF, 1 mM benzamidine, 1 mM Na₃VO₄, 1 mM DTT, 0.1%Brij-35). The homogenate was transported to the laboratory on ice andcentrifuged at 30,000 g for 1 h. The supernatant was cleared byultracentrifugation (50,000 g, 30 min), the pH adjusted to 6.8 andloaded onto a Büchner funnel containing 150 ml Whatman P11 equilibratedwith buffer B (50 mM MES pH 6.8, 2 mM EGTA, 50 mM NaF, 1 mM PMSF, 1 mMbenzamidine, 1 mM Na₃VO4, 1 mM DTT, 0.1% Brij-35), by applying a slightvacuum. The phosphocellulose was washed with 500 ml of buffer B andeluted stepwise with 150 ml each of buffer B containing 0.15 M -1 M NaCl(FIG. 2A). Fractions were screened for activity by phosphorylation of atau construct (K18) consisting of the four microtubule binding repeats,essentially as described (Drewes et al., 1992). Active fractions werefractionated by ammonium sulfate precipitation. The precipitate obtainedbetween 30 and 50 % saturation was dialysed against buffer A overnighton ice. The dialysate (approx. 50 ml) was cleared by ultracentrifugationand loaded onto an anion exchange column (Q-Sepharose HR, Pharmacia,80×16 mm) using a Superloop (Pharmacia) After washing the column with100 ml of buffer A and elution with a stepwise gradient from 0-0.5 MNaCl (FIG. 2B, flow rate 5 ml/min, fraction size 7 ml), active fractions(approx. 40 ml) were dialysed against buffer B and loaded onto a cationexchange column (SP-Sepharose HR, Pharmacia, 60×16 mm) (FIG. 2C, flowrate 4 ml/min, fraction size 7 ml). After elution with 0-0.5 M NaCl,active fractions (approx 40 ml) were pooled, the buffer was changed forbuffer A on a Sephadex G25 column (300×26 mm) and loaded onto a Mono QHR 5/5 column (Pharmacia) and eluted with a steep NaCl gradient (FIG.2D, flow rate 0.5 ml/min, fraction size 1 ml). Active fractions (2-3 ml)were concentrated twofold in a Centricon 30 microconcentrator (Amicon)and loaded onto a gel filtration column (Superdex 200, Pharmacia, 300×16mm) equilibrated and eluted with buffer A (pH 7.8, containing 150 mMNaCl and 10% glycerol).

[0139] The flow rate was 0.2 ml/min, fraction size was 2 ml. The columnhad previously been calibrated with a marker protein kit (Pharmacia).Active fractions were pooled, and the buffer was changed to buffer C (40mM β-glycerophosphate, 10 mM MgCl₂, 2 mM EGTA, 1 mM Benzamidine, 0.2 mMDTT, 0.1% Brij-35) on a Sephadex G25 column (100×16 mm). The proteinpool from the G25 column (10-15 ml) was loaded at 0.1 ml/min onto anATP-Sepharose column (Upstate Biotechnology Inc., Lake Placid, USA, 15×5mm). The column was washed with 5 ml of buffer C and eluted with 2 ml ofbuffer C containing 5 mM MgATP. The eluate was concentrated and freedfrom ATP and buffer substances on a Mono Q PC 1.6/5 column (‘Smart’system, Pharmacia), eluted with 25 mM Tris-HCl, pH 7.4, containing 250mM NaCl, 1 mM EGTA, 0.2 mM DTT, 1 mM benzamidine and 0.03% Brij-35.Active fractions were mixed with 50% (v/v) glycerol and stored at -20°C. Under these conditions, activity was preserved for at least onemonth.

[0140] With these six chromatographic steps used a ≈10,000 foldpurification of a Ser262-phosphorylating activity from a porcine braintissue extract was achieved. As shown in detail in FIG. 2,phosphocellulose (A), ion exchange chromatography on Q- and SP-Sepharoseand Mono Q (B,C,D), gel filtration (E) and, finally, affinitychromatography using immobilised ATP were employed. The activity of thiskinase in the tissue extract was ≈0.2 mU/mg, the activity of theaffinity-purified kinase ≈2 U/mg (1 unit transfers 1 Amol of phosphateper minute). The molecular weight of the enzyme was around 90-100 kDalby gel filtration, but the activity peak was broad and often showedpronounced tailing (FIG. 2E). On SDS gels, the apparent molecular weightwas ≈110 kDal (FIG. 3). The enzyme could be renatured in the gel; if tauwas polymerised into the gel matrix as a substrate and the gel wasincubated with γ-³²P-ATP, the 110 kDal band became prominent uponautoradiography (FIG. 3, lane 4-6), whereas some minor contaminationsobserved in the silver stained gel had no detectable activity. Afterphosphorylation with NPK-110, both whole tau and construct K18 showedsmall but distinct mobility change in SDS PAGE (FIG. 4, lanes 1-4). Thefinal amount of incorporated phosphate is ≈1.8- 2.5 mol per mole of tau,depending somewhat on enzyme concentration and activity; this level ofphosphorylation could be achieved after ≈2 hours. Phosphorylationreactions were carried out as follows:

[0141] Phosphorylation reactions were carried out in 40 mM Hepes, pH7.2, containing 2 mM ATP, 5 mM MgCl₂, 2 mM EGTA; 1 mM DTT, 0.1 mM PMSF,0.03% Brij-35. When extracts or crude fractions of kinase preparationswere screened, 50 mM NaF or 1 μM okadaic acid (LC Services, Woburn,Mass., USA) was included. Reactions were terminated by heating to 95° C.Phosphorylation was assayed in SDS gels (Steiner et al., 1990) or onphosphocellulose paper discs (Gibco) (Casnellie, 1991). In-gelphosphorylation assays were performed according to the method of Geahlenet al., 1986.

[0142] The specificity of NPK110 for tau was examined by trypticdigestion of phosphorylated protein and subsequent two-dimensional thinlayer electrophoresis and chromatography

[0143] (FIG. 5). If one compares the phosphorylation patterns obtainedfrom recombinant full-length 4-repeat tau (FIG. 5A) and the 4-repeatfragment K18 (FIG. 5B), it is apparent that most phosphorylated peptidesare generated from the repeat domain. This was confirmed by analysis ofa mixture of both samples (FIG. 5D). In a second approach, the trypticdigest was resolved by HPLC (not shown). In more detail, theseapproaches were carried out as follows:

[0144] Following phosphorylation reactions, the kinases were removed byboiling of the samples in 0.5 M NaCl/10 mM DTT and centrifugation. Tauprotein remains in the supernatant and was precipitated by 15% TCA.Cysteine residues were modified by performic acid treatment (Hirs,1967). The protein was digested overnight with trypsin (Promega,sequencing grade) in the presence of 0.1 mM CaCl2, using two additionsof the enzyme in a ratio of 1:10-1:20 (w/w) Two-dimensionalphosphopeptide mapping on thin layer cellulose plates (Macherey & Nagel,Düren, FRG) was performed according to Boyle et al., 1991. In brief,first dimension electrophoresis was carried out at pH 1.9 in formic acid(88%)/acetic acid/water (50/156/1794), second dimension chromatographyin n-butanol/pyridine/acetic acid/water (150/100/30/120). For themapping of phosphorylation sites by sequencing, recombinant human tau(200 μg, clone htau 40) was phosphorylated with NPK110 and ³²P-ATP (100Ci/mol) for 2 hours. The phosphorylation was terminated by a brief heattreatment. The protein was incubated with 6 M urea and 2 mM DTT, andcysteines were blocked with vinylpyridine (Tarr et al., 1983) orperformic acid treatment. After dialysis against 10 mM ammoniumbicarbonate, the protein was lyophilised and digested with trypsin(1:20) in the presence of 0.1 mM CaCl₂. Separation of peptides wasperformed by two successive HPLC runs on a μRPC C2/C18 SC 2/10 column(‘Smart’ system, Pharmacia). The digest was acidified with acetic acid(5% v/v) and fractionated by HPLC using a gradient of acetonitrile in 10mM ammoniumacetate (flow rate 0.1 ml/min, 0-25% in 120 min, 25-50% in 20min). Peptides were detected by UV absorption at 214, 254 and 280 nm andincorporated phosphate was measured as Cerenkov radiation in ascintillation counter (Hewlett-Packard TriCarb 1900 CA). Flowthroughfractions and radioactive peaks from this gradient were further purifiedusing a gradient of acetonitrile in TFA (flow rate 0.1 ml/min, 0%acetonitrile/0.075% TFA to 66% acetonitrile/0.05% TFA in 60 min).Sequence analysis of peptides was performed using a 477A pulsed liquidphase sequencer and a 120A online PTH amino acid analyser (AppliedBiosystems). Phosphoserines were identified as the dithiothreitol adductof dehydroalanine by gas phase sequencing (Meyer et al., 1991).

[0145] This yielded several labelled peptides which were analysed bydirect phosphopeptide sequencing and by phosphoamino acid analysis.Phosphoamino acid analysis: Aliquots of digestion samples were partiallyhydrolysed in 6N HCl (110° C., 60 min) and analysed by two dimensionalelectrophoresis at pH 1.9 and pH 3.5 (Boyle et al., 1991). The resultsof the phosphopeptide sequencing are compiled in Table 1. TABLE 1Tryptic phosphopeptides from htau40 phosphorylated with NPK110, obtainedby HPLC. The sequences are those of the main radioactive peaks. Listedare the number of counts obtained after the second purification run, theamount of material, the sequence with the phosphorylated residue(identified as S-ethylcysteine) starred, the phosphorylation site(numbering according to htau40). Note that the tryptic phosphopeptideCGSK from the second repeat was not detected by HPLC (presumably becauseof its small size and hydrophilicity) and thus had to be indentified byphosphopeptide mapping and site-directed mutagenesis. pmoles cpm inpeptide found Sequence found Phosph. sites 400.00 1000 IGS*TENLK Ser-262150.000 350 IGS*LDNIPHVPGGGNHK Ser-356 150.000 300 CGS*LGNIHHK Ser-32460,000 200 HVPGGGS*VQIVYK Ser-305

[0146] Most of the radioactivity was found in a peptide containingphosphorylated Ser262. Ser356 (in the KIGS motif of the fourth repeat)and Ser324 (from the KCGS motif of the third repeat) were also foundradioactively labelled. Two dimensional analysis of these purifiedpeptides lead to the identification of spots shown in FIG. 5C. Thisclearly shows that Ser262 (spot 1) is the main target site of NPK110 ontau, followed by Ser356 (spot 2). Spot 3 was identified as the peptidecontaining Ser305, spot 4 as Ser324 (in the KCGS motif of the thirdrepeat), spot 5 as Ser293 (in the KCGS motif of the second repeat). Thecorresponding tryptic peptide (²⁹¹CGSK) could not be isolated directlyby reverse phase HPLC chromatography, presumably because of itsshortness and hydrophilicity. It was therefore identified by sitedirected mutagenesis, using point mutants of K18 where the serines inall four KXGS motifs (Ser262, 293, 324, 356) were converted intoalanines. After phosphorylation with NPK110 only spot 3 (Ser305) wasvisible, while spots 1, 2, 4 and 5 were gone, thus identifying spot 5with Ser293 (data not shown).

EXAMPLE 2

[0147] Tau-microtubule binding and dynamic instability.

[0148] Previously it was shown that the phosphorylation of Ser262strongly decreased the interaction between tau and microtubules; thatis, not only the dissociation constant increased but also thestoichiometry decreased. Confirming these observations, a similar resultwas obtained after phosphorylation of tau by NPK110. In fact, FIG. GAshows that the reduction in binding is even more pronounced: NPK110completely abolishes microtubule binding within the concentration rangeaccessible. Because the binding became so weak it was also no longerpossible to estimate values for the dissociation constant and thestoichiometry. In other words, NPK110 efficiently causes the loss ofbinding of tau to microtubules. Binding studies were carried out asfollows:

[0149] Binding studies were performed by measuring co-sedimentation oftaxol-stabilised microtubules (30 μM) and tau by ultracentrifugation(Beckman TL 100) of 30 μl-samples. Aliquots of the pellet andsupernatant were assayed using SDS-PAGE and Coomassie blue staining.Scanner densitometry of dried gels was used for quantification ofprotein (for details see Gustke et al., 1992).

[0150] In order to verify this result a point mutation (Ser262 to Ala)was introduced into tau so that this site could no longer bephosphorylated. In this case, incubation of the mutant with NPK110 leftthe microtubule binding capacity largely intact, although there was somedecrease in affinity and stoichiometry (≈25%, FIG. 6A). This confirmstwo points of prior art studies, (i) phosphorylation of Ser262 is themajor switch controlling tau's affinity for microtubules, (ii) the othersites phosphorylated by the kinase have a small but measurable effect onthe binding (i.e. mainly the equivalent serines in the KXGS motifs ofrepeats 2, 3, and 4).

[0151] The next question was: Do microtubules protect tau from beingphosphorylated by NPK110? If this were the case, then tau —once bound tomicrotubules— might retain its high affinity for microtubules. To answerthis point, taxol-stabilised stabilised microtubules were firstsaturated with tau, and then incubated with NPK110. As illustrated inFIG. 6B, tau gradually dissociates from microtubules, concomitant withphosphorylation. Thus microtubules retard phosphorylation of tau by thekinase but cannot prevent it.

[0152] One important function of tau is to stabilise microtubules andsuppress their dynamic instability (Drechsel et al., 1992). Thus, if tauloses its binding to microtubules one would expect stable microtubulesto become dynamic. This effect can be illustrated by video dark fieldmicroscopy of individual microtubules seeded onto flagellar axonemes(FIG. 7). The experiment was carried out as follows:

[0153] Video microscopy of microtubules nucleated on axonemes was doneessentially as described (Trinczek et al., 1993). Briefly, 5 μMPC-tubulin, 2.5 μM tau (unphosphorylated or phosphorylated) and lowamounts of sea urchin sperm axonemes (10-100 fM) were mixed in 50 m MNa-Pipes, pH 6.9, containing 3 mM MgCl₂, 2 mM EGTA, 1 mM GTP and 1 mMDTT. 1.0 μl of the samples was put on a slide, covered with 18 ×18 mmcoverslips, sealed, and warmed up to 37° C. in a temperature-controlledair flow within 5 s. A constant temperature of 37° C. was maintained bythe air flow. The axoneme nucleated microtubules were recorded at time2.5, 5, 10, 15, 20, 25, and 30 min after the temperature shift. For eachcondition and time three to five axonemes of a sample and 10-20experiments were analysed, and the lengths of 500-600 microtubule plusends were measured. Only those microtubules which were clearly locatedwithin the focal plane were taken into account. The depth of solutionwas 3-4 μm, and the focal depth was 1-2 μm.

[0154] In the experiment of FIG. 8A the concentration of tubulin (5μM)was chosen such that microtubules would not assemble by themselves butwould grow upon addition of (unphosphorylated) tau. Tau phosphorylatedwith NPK110 did not support growth whereas the mutant Ser262-Ala did. Inother words, tau phosphorylated at Ser262 behaved as “no tau” because itdid not interact with microtubules, in contrast to the mutant which did.Even more dramatic is the conversion of microtubules from undynamic todynamic behaviour under the influence of the kinase. In the experimentof FIG. 8B microtubules were allowed to grow off axonemes in thepresence of tau and their mean length which increased to ≈50 μm over 20min was recorded. In a parallel experiment NPK110 with ATP was added (orwithout ATP as a control). In the control experiment (without ATP)microtubules were able to grow continuously and showed little dynamicinstability (FIG. 8B, open circles). With ATP added, the mean lengthincreased only to 20μm and then dropped again, due to the gradualphosphorylation of tau and concomitant increase in microtubule dynamics(filled circles). When the mutant Ser262-Ala was used, microtubules grewnormally even when the kinase and ATP were present (triangles). Theseresults are summarised in the length histograms of FIG. 8C-D. At earlytimes after initiation of assembly microtubules are short and ratherhomogeneous in length (peaks of open circles at 5 min), at later timesof uninterrupted growth the microtubules become long and show a broadlength distribution (filled circles in FIGS. 8C and 8E). However, whenthe kinase is allowed to phosphorylate Ser262 (i.e. the kinase, ATP, andwild type tau with Ser262 are present), microtubules remain short (opencircles in FIG. 8D).

EXAMPLE 3

[0155] Other kinases phosphorylating the repeat domain of tau. Tau canbe phosphorylated in vitro by many kinases which can be classified byseveral criteria, depending on function, targets, or others. Certainproline-directed kinases that are of diagnostic interest for Alzheimer'sdisease (because of the antibody reactions induced by them)phosphorylate the regions flanking the repeats but appear to have littleinfluence on tau-microtubule binding. Conversely, one would expect thatkinases phosphorylating the repeat region have an influence onmicrotubule binding because the repeats of tau are thought to beinvolved in this function, and this is in fact borne out by the resultswith NPK110 described so far. The question therefore arises how thiskinase compares with other kinases phosphorylating tau in the repeatdomain. Several of these have been reported so far (Table 2). TABLE 2Summary of phosphorylation sites and kinases affecting the repeats andnearby regions of tau (only non-proline directed kinases and sites arelisted). Major sites are denoted by X, minor ones by (x). Note that theresults were obtained by different methods: (1) phosphorylation of taufollowed by proteolytic digestion, separation of peptides, andphosphopeptide sequencing (Steiner et al., 1990, Steiner, 1993, Gustkeet al., 1992; Scott et al., 1993). (2) Mass spectrometry ofphosphopeptides combined with sequencing (Hasegawa et al., 1992;Watanabe et al., 1993). (3) Phosphorylation of a synthetic peptide(Correas et al., 1992). (4) 2D mapping of phosphopeptides combined withsequencing (this report). Since these data are derived from the repeatdomain K18 they do not contain information on possible phosphorylationsites outside the repeats. kinase or reference S262 S293 S324 S356activity S214 KIGS KCGS S305 KCGS KIGS S377 S409 S416 PKA Scott et al.,(x) (x) (x) X X 1993 PKA Steiner, 1993 X (x) (x) (x) X X PKA this reportND (x) (x) (x) X X ND ND PKC Correas et al., X 1992 PKC Steiner, 1993 X(x) (x) (x) X PKC this report ND (x) (x) (x) ND CaMK Steiner et al., X1990 brain ex. Gustke et al., X X 1992 brain ex. this report X X PK35/41 Biernat et al., X (x) (x) X 1993 NPK110 this report X (x) (x) (x)X brain in vivo: Alzheimer: Hasegawa et al., X 1992 adult: Watanabe etal., - - - no sites in repeat region - - - 1993 fetal: Watanabe etal., - - - no sites in repeat region - - - 1993

[0156] For example, PKA phosphorylates mainly Ser214, Ser409 and Ser416outside the repeats, but minor sites include Ser324 and Ser356 withinthe repeats (Scott et al., 1993; Steiner, 1993). Since Ser262 is not oneof the sites one would not expect a major effect on microtule binding,in agreement with our observations. PKC sites include the KCGS motif inrepeat 3 (Correas et al., 1992; Steiner, 1993), again with no majoreffect on microtubule binding in our hands. The partially purifiedkinase activity described previously (Biernat et al., 1993)phosphorylated all four KXGS motifs, and finally, the kinase activitiesfrom brain extract phosphorylated both the Ser/Thr-Pro motifs as well asSer262 and Ser356 (Gustke et al., 1992), with the reported strongeffects on microtubule binding due to Ser262. The strategy employed inthese studies was to generate proteolytic fragments from phosphorylatedtau which were then separated by HPLC and identified by sequencing. Thisusually generates a multitude of peptides whose recovery is not alwayslinear, making it difficult to judge the relative amount ofphosphorylation at different sites.

[0157] Because of these uncertainties it was decided to re-investigatethe phosphorylation sites by a different approach. The phosphopeptideswere analysed not only by HPLC and sequencing, but also bytwo-dimensional mapping on thin layer cellulose plates which gives aclearer representation of the relative contributions. Full length4-repeat tau and the repeat domain (K18) were phosphorylated with brainextract, NPK110, PKC, and PKA. This enabled the comparison of thephosphorylation sites in the repeat domain of tau and showed the extentof this phosphorylation in htau40 by each of the kinases. The resultsare shown in FIG. 9 where the phosphopeptides derived from K18 arelabelled according to FIG. 5. Phosphopeptide spots generated by theother kinases were identified by running each sample along with the K18sample phosphorylated with NPK110 (data not shown).

[0158] The patterns shown in FIG. 9A were obtained by phosphorylatingfull length tau and K18 with brain extract. With full length tau onlyspot 1 (Ser262) is clearly seen, spot 2 (Ser356) is barely visible. Thisis even more prominent in the phosphorylation pattern of K18.

[0159] When the phosphorylation of K18 by NPK110were examined, a peptidepattern similar to that of the brain extract (compare FIGS. 9A and 9B)is formed; the most prominent spots are 1 and 2, containing Ser262 andSer356, while Ser 305 (spot 3), Ser324 (spot 4), and Ser293 (spot 5)represent minor components. This confirms the role of NPK110 as themajor Ser262 kinase. By contrast, re-investigation of the earlier kinaseactivity (Biernat et al., 1993) has so far yielded inhomogeneousresults. Although it phosphorylates the same serines as NPK110 theweighting is different, and the activity of the kinase in brain extractis at least 10-fold lower. This explains why even long incubations oftau with this kinase activity lead to only partial suppression of tau'sbinding to microtubules, as described earlier.

[0160] As seen in FIG. 9C, PKC only phosphorylated Ser305 (spot 3),Ser324 (spot 4) and Ser293 (spot 5) to a significant extent in K18. Thesmear and the outermost spot to the left (arrow) are not phosphopeptidesderived from tau since they also occurred in control experiments whereno tau construct had been added (not shown). The remaining two spotscould not be identified; the spot on the upper right (starred) did notcolocalise with either Ser262 (spot 1) or Ser356 (spot 2). Comparison ofthis pattern with the one obtained from full length tau revealed thatthe major phosphorylation sites of PKC are outside the repeat domain.Only Ser305 (spot 3) was faintly visible in this pattern (note that thespot on the upper right does not correspond to the upper right spot fromK18 (starred), as confirmed by control experiments (not shown)).

[0161] When using purified PKA to phosphorylate full length tau andconstruct K18 (FIG. 9D) mainly Ser356 (spot 2), Ser305 (spot 3), Ser 324(spot 4) and Ser293 (spot 5) are found. Ser262 (spot 1) is only a minorphosphorylation site. Phosphorylation of full length tau (FIG. 9D, leftpanel) yielded similar spots, plus additional sites outside the repeatregion of tau. These result are in general agreement with earlier data(Scott et al., 1993; Steiner, 1993). Some of these sites had also beenseen with the “35/41 kDal” kinase activity described previously (Biernatet al., 1993). In subsequent experiments it was determined that the 41kD component is the catalytic subunit of PKA (using an antibody againstPKA obtained from H. Hilz, Hamburg, data not shown); this explains inpart the overlap in the data. PKC phosphorylates mainly Ser305, Ser293and Ser324 (the latter in agreement with Correas et al., 1992), but notSer262 (FIG. 9C).

EXAMPLE 4

[0162] Sites of MAP2 and MAP4 phosphorylated by the kinase NPK110. MAP2and MAP4 are two microtubule-associated proteins which belong to thesame MAP-family as tau because they show high homology in the region ofthe 3 or 4 internal repeats where the proteins bind to microtubules (forreview see Chapin & Bulinski, 1992). MAP2 occurs preferentially inbrain, mostly in the somatodendritic compartment of neurons. Like tau,MAP2 can be expressed in different forms due to alternative splicing(Kindler et al., 1990): The second repeat may be absent (this is the“classical” MAP2); in addition the region of residues 152-1514 (i.e.1363 out of 1830 residues) may be absent (generating a protein with 467residues; this form is commonly called MAP2c). The phosphorylationexperiments described here have been performed with recombinant MAP2cexpressed in E. coli (Table 3). TABLE 3 Peaks from Peptide second no.extinction peptide phosphor. col. (FIG. 11) cpm (214 nm) sequenceresidue 1 I 300,000 0.05 1705.CGS*LK Ser1707 (in 2nd repeat) 2 II200,000 0.3 1674:IGS* Ser1676 TDNIK (in 1st repeat) 3 III 100,000 0.833:DQGGS Ser 37 GEGLSR Ser1536 1535:SS*LPP 4 IV 100,000 0.61791:LS*NVSS* Ser1792 SGS*IN Ser1796 Ser1799

[0163] MAP4 is a ubiquitous MAP which is probably involved in mitosis,it also occurs as several splicing isoforms (West et al., 1991). Thephosphorylation experiments have been done with a recombinant MAP4construct comprising the C-terminal 496 residues (including the repeatdomain) and expresssed in E. coli (Table 4). TABLE 4 Peaks from Peptidesecond no. extinction peptide phosphor. col. (FIG. 12) cpm (214 nm)sequence residue 1 I 135,000 0.8 825:SPATT*LP Thr829 2 II 150,000 0.35939:VGS* Ser941 TENIK (in 1st repeat) 3 III 120,000 0.35 923:LATTVS*Ser928 APDLK 4 IV 100,000 0.8 872:NT*T*PT* Thr873 GAAPP Thr874 Thr876 5V 55,000 0.3 898:SS*GALS* Ser899 VDK Ser903 6 VI 100,000 0.8 1071:VGS*LDSer1073 (in 4th repeat) 7 VII 33,000 0.04 923:LATTVS* Ser928 APDLK

[0164] The Phosphorylation methods are identical to the ones describedin Example 2. MAP2 and MAP4 were phosphorylated with NPK110 usingradioactive ATP, the phosphorylated protein was digested with trypsinand analysed by two-dimensional phosphopeptide mapping (FIG. 11 forMAP2c, FIG. 12 for MAP4 construct). The peptides were then purified bytwo HPLC gradient columns. The purified radioactive peptides weresequenced (for identification of the phoshorylated residues) andidentified by two-dimensional phosphopeptide mapping.

[0165] Effects of phosphorylation on interactions with microtubules:

[0166] The effects of phosphorylation of MAP2 and MAP4 by NPK110 werethe same as for tau, that is, the affinity to microtubules decreasedseveral-fold, and the dynamic instability of microtubules became muchgreater. This can be demonstrated, for example, by the decrease in themean length of microtubules in the presence of the MAP in question, thekinase NPK110, and ATP (required for phosphorylation). FIG. 13 showsexamples for the cases of MAP4, MAP2, and MAP2c. Microtubule assemblystarts at time 0. Hollow circles show the increase of mean length in theabsence of ATP (no phosphorylation). Filled circles show that in thepresence of ATP (and therefore with phosphorylated MAPs) the mean lengthis only about half of the control.

[0167] The biological significance of the novel NPK-110 can besummarised as followed:

[0168] NPK-110 is an efficient kinase for the repeat domain of tau,MAP2, MAP2c and MAP4. It phosphorylates all four KXGS motifs in tau, thefirst and fourth (Ser262 and Ser356) being the most pronounced sites. Inthis regard the kinase reproduces earlier observations with the kinaseactivity from the brain extract (Gustke et al., 1992, and see FIG. 9).The most dramatic effects of the kinase are that it virtually eliminatestau's binding to microtubules (FIG. 6B), it causes the release of taufrom microtubules, and it turns stable microtubules into dynamicallyunstable ones, as seen by video microscopy. These effects are mainlydependent on the phosphorylation of Ser262, as shown by the point mutantSer262-Ala. These features make NPK110 a candidate enzyme forcontrolling the state of assembly of microtubules in neurons. They arealso consistent with the “Tau Hypothesis of Alzheimer's Disease” whichassumes that tau's failure to bind to and stabilise microtubules leadsto their breakdown and cessation of axonal transport. This could occureither by the detachment of tau from microtubules, or by the inhibitionof newly synthesised tau to bind to microtubules, in both casesresulting from phosphorylation. According to this scheme, anintervention that would slow down NPK110 or turn off its potentialactivating cascade would be suitable for a treatment of Alzheimer'sdisease.

[0169] It is furthermore noted that the motif KXGS is conserved not onlywithin the tau repeats, but also within other MAPs such as the neuronalMAP2 and the ubiquitous MAP4 (for review see Chapin & Bulinski, 1992).It is therefore possible that NPK-110 has a more general role, affectingdifferent MAPs and perhaps other proteins. One role which might beenvisaged is the involvement of NPK-110 in the generation of cancer.

EXAMPLE 5 Further Characterization of the NPK of the InventionDescription of the cDNA Clones

[0170] A screening of a rat brain cDNA library with degenerateoligonucleotides derived from the brain-p110MARK peptide sequencesyielded nine clones which were sequenced. They code for at least twodifferent kinases from at least two different genes, with a 70% mutualhomology. The peptide sequences fit completely with the larger clone,termed MARK-1 (corresponding to NPK-110), whose 5′-prime end is missing(mol. wt. of the encoded protein approx. 90 kDal). The smaller cDNAMARK-2 encodes a protein of 81 kDal. Peptides suitable for the design ofoligonucleotides for screening said cDNA libraries is provided in Table5. The amino acid sequences of the identified clones are provided inTable 6.

Homologies

[0171] A database search for homologous sequences obtained two relatedbut no identical sequences:

[0172] MMKEM (X70764), a mouse CDNA encoding a putative protein kinaseof unknown function (Inglis et al., 1993), shows 73% homology to MARK-1and 96% homology to MARK2.

[0173] HUMP78A (M80359), an unpublished human cDNA sequence, shows 73%homology to MARK-1 and 69% homology to MARK-2. All kinases show a lowhomology (about 25%) to the KIN1 and KIN2 proteins from Saccharomycescerevisiae (Levin et al., 1987, 1990).

Tissue Distribution

[0174] As judged by Northern blotting (FIG. 14), MARK-1 and MARK-2 mRNAsare ubiquitously expressed in fetal and adult tissues. Expression ishighest in muscle, brain and fetal (but not adult) kidney.

Activation

[0175] p110/MARK prepared from brain is at least 100-fold more activethan MARK expressed in E. Coli. The activity is dependent onphosporylation of MARK itself on Ser and/or Thr residues, since, afterdephosphorylation with phosphatase 2A, all activity is lost.

[0176] The phosphorylation of pllO/MARK reveals an apparent molecularweight of 110kD on SDS gels, whereas the predicted molecular weight fromcDNA sequencing is 90 kD. This shift in apparent molecular weight isoften observed -with phosphoproteins.

Targets

[0177] p110MARK phosphorylates not only tau protein, but also relatedMAPs such as MAP2 or MAP2c (neuronal MAPs largely confined to thesomatodendritic compartment) and MAP4 (a ubiquitous MAP), indicating awidespread function of the enzyme. The major phosphorylation sites aresimilar in these MAPs, namely the serines in the KXGS motifs in therepeat domain. The effect of phosphorylation is also comparable, namelya strong reduction in the microtubule-binding capacity of the MAPs, andhence a loss of microtubule stability (see FIGS. 15, 16 for examples).TABLE 5 Peptide sequences obtained from a porcine brain MARK preparationby lysC digestion. Fraction Sequence: 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 18  33-12 D R W M N V G H E  E  E  E  L  K  P  Y  A  E 19 20 21 P  E  P  41 I A N E L K  47-16 A E N L L L D A D  M  N  I  K  71-09* XS S R Q N I P R  C  R  N  N  I  I  85 I L N H P N I V K  87-24* L D T FC G S P P  Y  A  A  P  E  L  F  Q  G  K 120 L F V L N P I K 121 L F R EV R I X 130-13 Y R I P F Y M S T  D  C  E  N 140-9 F R Q I V S A V Q  Y C  H  Q  K 140-20 R I E I M V T M G  F  L

[0178]

1. A DNA sequence encoding a neuronal protein kinase (NPK) or afunctional fragment thereof that is capable of phosphorylating asequence motive of the type KXGS in tau, MAP4, MAP2 and MAP2ccharacterised by the following features: (a) it encodes the amino acidsequence depicted as MARK-1 in Table 6; (b) it encodes the amino acidsequence depicted as MARK-2 in Table 6; or (c) it hybridises to the DNAof (a) or (b).
 2. The DNA sequence according to claim 1, wherein the NPKis further characterised by the following features: (a) it has anapparent molecular weight of 110 kD as determined by SDS-PAGE; (b) itphosphorylates Serine residues 262, 293, 305, 324 and 356 of human tauprotein; and (c) it comprises the following amino acid sequencesKLDTFCGSPPYAAPELFQGK DRWMNVGHEEEELKPYAEP (K)SSRQNIPRCRNNI


3. The DNA sequence according to claim 1 or 2, wherein the NPK isfurther characterised by the following features: (d) it is deactivatedby phosphatases PP-2A; and (e) it phosphorylates the following Serine orThreonine residues of tau related microtubule associated proteins (MAPs)MAP2, MAP2c and MAP4 MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796,S1799 MAP4: T829, T873, T874, T876, S899, S903, S92B, S941, S1073 (f) itcauses the dissociation of tau, MAP4, MAP2 and MAP2c from microtubules4. The DNA sequence according to any one of claims 1 to 3, wherein theNPK is obtainable from brain tissue by the following steps: (a)homogenisation of brain extract and subsequent centrifugation thereof;(b) chromatography of the supernatant obtained in step (a) oncellulosephosphate wherein the NPK active fractions elute between 200 to400 mM NaCl; (c) ammonium sulfate precipitation of active fractionsobtained in step (b) and dialyses of the precipitate; (d) anion exchangechromatography of the dialysate obtained in step (c) on Q-Sepharose(Pharmacia) and elution of the NPK active fractions wherein said NPKactive fractions elute as a single peak at about 0.2 M NaCl, withsubsequent dialyses of the active fractions; (e) cation exchangechromatography on Mono S HR 10/10 (Pharmacia); (f) chromatography onMono Q HR 5/5, wherein the NPK active fractions elute at about 250 mMNaCl; (g) gel filtration chromatography on Superdex G-200, wherein theNPK activity elutes with an apparent molecular weight of 100 kD; and (h)affinity chromatography on ATP-cellulose, wherein the NPK activefractions elute with an apparent molecular weight of about 110 kD asdetermined by SDS-PAGE; wherein the NPK activity is measured byincubating a peptide comprising amino acid residues 255 to 267 of humanadult tau in the presence of radioactively labelled ATP and determiningthe radioactivity incorporated into said peptide.
 5. The DNA sequenceaccording to any one of claims 1 to 4, wherein the NPK is an NPK from amammalian brain.
 6. The DNA according to claim 5, wherein said mammalianbrain is human or porcine brain.
 7. A polypeptide encoded by the DNAsequence of any one of claims 1 to 6, or a functional fragment thereof.8. A Serine or Threonine residue phosphorylated by the polypeptide ofclaim 7, said Serine or Threonine residue being located in the followingamino acid position of tau related microtubule associated proteins(MAPs) MAP2, MAP2c and MAP4: MAP2/MAP2C: S37, S1536, S1676, S1707,S1792, S1796, S1799 MAP4: T829, T873, T874, T876, S899, S903, S928,S941, S1073
 9. An epitope comprising the Serine or Threonine residue ofclaim
 8. 10. An antibody specifically binding to the polypeptide orfragment thereof according to claim
 7. 11. An antibody specificallybinding to the epitope of claim
 9. 12. The antibody according to claim10 or 11, which is a monoclonal antibody or a derivative or fragmentthereof.
 13. The antibody according to claim 10 or 11, which is apolyclonal antibody or a derivative or fragment thereof.
 14. Apharmaceutical composition which is containing a specific inhibitor forthe polypeptide or fragment thereof according to claim 7, optionally incombination with a pharmaceutically acceptable carrier and/or diluent.15. The pharmaceutical composition according to claim 14 for thetreatment of Alzheimer's disease.
 16. The pharmaceutical compositionaccording to claim 14 for the treatment of cancer.
 17. Thepharmaceutical composition according to any one of claims 11 to 13wherein said inhibitor is the antibody according to any one of claims 10to 13, a phosphatase capable of dephosphorylating the polypeptide orfragment thereof according to claim 7, preferably phosphatase PP-2A, aninhibitor of the activating kinase of the polypeptide of claim 7, a tauderived peptide comprising the Ser262 residue, or a MAP4 or MAP2/MAP2cderived peptide comprising at least one of Serine or Threonine residuesmentioned in claim
 8. 18. A diagnostic composition comprising: (a) thepolypeptide according to claim 7; (b) the antibody according to any oneof claims 10 to 13; and/or (c) a peptide comprising the serine residueaccording to claim 2(e).
 19. A method for the in vitro diagnosis and/ormonitoring of Alzheimer's disease comprising assaying a cerebrospinalfluid isolate of patient or carrying out a biopsy of nerve tissue (forexample, olfactory epithilium) and testing said tissue for the presenceof the polypeptide or fragment thereof according to claim
 7. 20. Amethod for the in vitro diagnosis and/or monitoring of Alzheimer'sdisease comprising assaying a cerebrospinal fluid isolate of a patientor carrying out a biopsy of nerve tissue and testing said tissue for thepresence of unphysiological amounts or activity of the polypeptide orfragment thereof according to claim
 7. 21. The method according to claim19 or 20, wherein the NPK is detected by the antibody according to anyone of claims 10, 12 or
 13. 22. A method for the in vitro diagnosis forcancer or the onset of cancer comprising assaying a suitable tissue orbody fluid for the presence of phosphorylated Serine or Threonineresidues of tau related microtubule associated proteins (MAPs) MAP2,MAP2c and MAP4 in the positions: MAP2/MAP2c: S37, S1536, S1676, S1707,S1792, S1796, S1799 MAP4: T829, T873, T874, T876, S899, S903, S928,S941, S1073 or for the presence of unphysiological amounts of thepolypeptide or fragment of claim 7 or an specific phosphatase for saidpolypeptide or fragment.
 23. A method for the in vitro conversion ofnormal MAP2, MAP2c or MAP4 by the treatment with the polypeptide orfragment of claim 7 into proteins phosphorylated at positions:MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796, S1799 MAP4: T829,T873, T874, T876, S899, S903, S928, S941, S1073 said phosphorylationstatus being indicative of cancer or the onset of cancer.
 24. Use of thephosphorylated Serine or Threonine residue(s) of the MAP of claim 8 orthe epitope comprising said residue(s) of claim 9 for the generation ofspecific antibodies indicative of cancer or the onset of cancer.
 25. AnRNA sequence complementary to the DNA sequence of any one of claims 1 to6.